The isolation and characterization of mutant cells unable to perform the various steps of endocytosis may be the only way to fully understand this complex series of events and the interrelationship of endocytosis of intracellular membrane trafficking. Mutants in Chinese hamster ovary cells which have been isolated seem to fall into two general types, either receptor minus or those defective in endosomal acidification. The pleotropic phenotype of the acidification mutation has already provided new insights into the common mechanism involved in membrane trafficking. Recently, we have isolated a subline of the human derived hepatoblastoma cell line, HepG2, resistant to an asialoglycoprotein conjugate of amethopterin. Both parental and mutant cell lines were equally sensitive to amethopterin alone, indicating that drug resistance was not responsible for the survival of the mutant. Analysis of the status of the asialoglycoprotein receptor content and function in these cells indicated that there was a 60% loss of receptor activity with no concomitant loss of receptor protein expression. It is reasonable to assume that mutations other than of these two general classes would be lethal to the cell, and, therefore, we propose to isolate conditional mutants for various steps in the pathway. To target the selection for such mutants, it is proposed to use two cytotoxic conjugates as selective agents. By using two unrelated receptors that undergo endocytosis, it should be possible to avoid the selection of mutants that are receptor-specific. Once isolated, the defect in the intracellular pathway will be defined at both a functional and genetic level. The long-term objective of this proposal is to provide insight into the biochemical elements which control the endocytotic pathway.